The present invention relates to the field of diagnosing and treating conditions related to apoptosis, or programmed cell death. More specifically, it relates to the identification and characterization of a novel gene family, the expression of which is associated with apoptosis.
Apoptosis is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death can be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation and infection by human immunodeficiency virus (HIV). Wyllie (1980) Nature 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun. 118:392-399; Duke and Cohen (1986) Lymphokine Res. 5:289-299; Tomei et al. (1988) Biochem. Biophys. Res. Commun. 155:324-331; Kruman et al. (1991) J. Cell. Physiol. 148:267-273; Ameisen and Capron (1991) Immunology Today 12:102; and Sheppard and Ascher (1992) J. AIDS 5:143. Agents that modulate the biological control of apoptosis thus have therapeutic utility in a wide variety of conditions.
Apoptotic cell death is characterized by cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage. Kerr et al. (1992) FASEB J. 6:2450; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267. The blebs, small, membrane-encapsulated spheres that pinch off of the surface of apoptotic cells, may continue to produce superoxide radicals which damage surrounding cell tissue and may be involved in inflammatory processes.
While apoptosis is a normal cellular event, it can also be induced by pathological conditions and a variety of injuries. Apoptosis is involved in a wide variety of conditions including, but not limited to, cardiovascular disease; cancer regression; immunoregulation; viral diseases; anemia; neurological disorders; gastrointestinal disorders, including but not limited to, diarrhea and dysentery; diabetes; hair loss; rejection of organ transplants; prostate hypertrophy; obesity; ocular disorders; stress; and aging.
Genes which have been shown to activate the apoptosis pathway in tumor cells include the FAS antigen, TNFxcex1 and TNFxcex2. See, e.g, Tomei and Cope et al. in Apoptosis II: The Molecular Basis of Apoptosis in Disease (1994) Cold Spring Harbor Laboratory Press. In the nematode C. elegans, mutations in the genes ced-3 and ced-4 prevent autonomous cell death during development. Yuan and Horvitz (1990) Dev. Biol. 138:33. A mutation which activates the nematode gene ced-9 prevents cell death during development, whereas mutations that inactive this gene promote programmed cell death. In mammalian cells, the p-53 gene has been shown to induce apoptosis in some cells, but not others.
Apoptosis-inhibiting genes under investigation include bcl-2 which was isolated from B-cell lymphomas and blocks apoptosis without affecting cell proliferation. See, e.g., Tsujimoto et al. Science 226:1087; Hockenberry et al. (1990) Nature 348:334. The mechanism by which bcl-2 inhibits apoptosis is not known. Mcl-1, expressed in myeloid cells, exhibits sequence similarity to bcl-2 and is believed to be involved in regulating apoptosis. Kozopas et al. (1993) Proc. Natl. Acad. Sci. USA 90:3516.
Members of a large family of putative transmembrane receptors related to the Drosophila melanogaster tissue polarity gene frizzled have been cloned recently. See, Wang et al. (1995) J. Biol. Chem. 271:4468. Frizzled family members are found in organisms as diverse as nematodes and humans and are expressed in a variety of tissues and during embryonic development. In Drosophila, frizzled mutations affect the polarity of structures, such as sensory bristles, on the body surface. The precise functions and clinical significance of the frizzled family in other species remains largely unknown.
All references cited herein, both supra and infra, are hereby incorporated by reference herein.
The present invention encompasses isolated polynucleotides, polypeptides and antibodies derived from or reactive with the products of the novel apoptosis-related genes. The invention also encompasses uses of these compositions.
Accordingly, one aspect of the invention is polynucleotides encoding polypeptides of the SARP family. Representative polypeptides are those having the amino acid sequence of SEQ. ID. NO: 2, 4, 6 or 7. The invention likewise encompasses polynucleotides encoding peptides having substantial homology to the amino acid sequence of SEQ. ID. NO: 2, 4, 6 or 7.
In another aspect, the invention provides isolated polynucleotides that are comprised of a region of at least 15 contiguous nucleotides, where these nucleotides are capable of forming a stable duplex with a polynucleotide encoding sequence of SEQ. ID. NO: 1, 3, 5 or 18.
Another aspect of the invention is cloning and expression vectors comprising the polynucleotides of the invention. Also included are host cells comprising the polynucleotides of the invention.
In another aspect, the invention comprises polypeptides of at least 11 amino acid residues of SEQ. ID. NO: 2, 4, 6 or 7 and further comprises polypeptides substantially homologous to 11 amino acid residues of SEQ. ID. NO: 2, 4, 6 or 7. The invention also provides fusion polypeptides comprising a polypeptide of the present invention.
The invention also provides for polyclonal or monoclonal antibodies which specifically bind to the polypeptides of the invention. There are termed xcex1SARP antibodies.
In another aspect, methods of detecting the polynucleotides of the invention are provided. These methods comprise contacting a biological sample under conditions that permit the formation of a stable complex, and detecting any stable complexes formed.
Another aspect of the invention is methods of detecting the SARP family of proteins. These methods entail the steps of contacting a biological sample obtained from an individual with an xcex1SARP antibody of the invention under conditions that permit the stable antigen-antibody complex and detecting stable complex formed, if any.
Also provided are methods for treatment of apoptosis by administration of a therapeutically effective amount of the polynucleotides and/or polypeptides of the invention to a patient in need of such treatment. The methods include making a composition for treatment of conditions related to apoptosis. Other methods using these compositions include preventing apoptosis in cultured cells, methods of increasing organ preservation for subsequent organ transplantation and in situ preservation for bypass operations, e.g., heart, liver, lungs, brain, etc., and methods of treating dermatological conditions in which apoptosis is implicated.
Also provided are methods for the detection of disease by providing a test sample of bodily fluid; assaying the test sample for the presence of a gene product of an hsarp gene; and comparing the amount of gene product detected in the test sample to the amount of gene product detected in a non-diseased sample of the same tissue type as the test sample. Assaying encompasses, but is not limited to, nucleic acid hybridization and antibodyxe2x80x94antigen interactions.
In an additional embodiment of the present invention, a method of treatment of a patient is provided, comprising administering to the patient a therapeutically effective amount of a pharmaceutically acceptable composition comprising a component selected from the group comprising a sarp or antisense-hsarp polynucleotide or a SARP polypeptide or SARP antibody. The method can be a method of treating apoptosis related conditions. In a specific embodiment, the patient is suffering from a condition related to cancer, including, but not limited to, cancer of the mammary tissue, the prostate or the prostate epithelial tissue. In an additional embodiment, the composition contains a sarp polynucleotide or the gene product of that polynucleotide, a SARP polypeptide.
The above and other objects of the invention will become readily apparent to those of skill in the relevant art from the following detailed description and figures, wherein only the preferred embodiments of the invention are shown and described, simply by way of illustration of the best mode of carrying out the invention. As is readily recognized, the invention is capable of modifications within the skill of the relevant art without departing from the spirit and scope of the invention.